Feature quality in array fabrication

ABSTRACT

A method, and apparatus and computer program products for executing the method. The method includes obtaining a set of multiple images of a target feature location on an array of multiple features, each image of the set representing the target feature location following deposition of a corresponding sub-set of multiple droplets for that feature. An overlay composite may be generated from the image set. The overlay composite may be used as a quality control tool, or in interrogating the array or processing results of the interrogation.

FIELD OF THE INVENTION

[0001] This invention relates to arrays, particularly polynucleotidearrays such as DNA arrays, which are useful in diagnostic, screening,gene expression analysis, and other applications.

BACKGROUND OF THE INVENTION

[0002] Polynucleotide arrays (such as DNA or RNA arrays), are known andare used, for example, as diagnostic or screening tools. Such arraysinclude regions of usually different sequence polynucleotides arrangedin a predetermined configuration on a substrate. These regions(sometimes referenced as “features”) are positioned at respectivelocations (“addresses”) on the substrate. The arrays, when exposed to asample, will exhibit an observed binding pattern. This binding patterncan be detected upon interrogating the array. For example allpolynucleotide targets (for example, DNA) in the sample can be labeledwith a suitable label (such as a fluorescent compound), and thefluorescence pattern on the array accurately observed following exposureto the sample. Assuming that the different sequence polynucleotides werecorrectly deposited in accordance with the predetermined configuration,then the observed binding pattern will be indicative of the presenceand/or concentration of one or more polynucleotide components of thesample.

[0003] Biopolymer arrays can be fabricated by depositing previouslyobtained biopolymers (such as from synthesis or natural sources) onto asubstrate, or by in situ synthesis methods. Methods of depositingobtained biopolymers include loading then touching a pin or capillary toa surface, such as described in U.S. Pat. No. 5,807,522 or deposition byfiring from a pulse jet such as an inkjet head, such as described in PCTpublications WO 95/25116 and WO 98/41531, and elsewhere. For in situfabrication methods, multiple different reagent droplets are depositedat a given target location in order to form the final feature (hence aprobe of the feature is synthesized on the array stubstrate). The insitu fabrication methods include those described in U.S. Pat. No.5,449,754 for synthesizing peptide arrays, and described in WO 98/41531and the references cited therein for polynucleotides. The in situ methodfor fabricating a polynucleotide array typically follows, at each of themultiple different addresses at which features are to be formed, thesame conventional iterative sequence used in forming polynucleotidesfrom nucleoside reagents on a support by means of known chemistry. Thisiterative sequence is as follows: (a) coupling a selected nucleosidethrough a phosphite linkage to a functionalized support in the firstiteration, or a nucleoside bound to the substrate (i.e. thenucleoside-modified substrate) in subsequent iterations; (b) optionally,but preferably, blocking unreacted hydroxyl groups on the substratebound nucleoside; (c) oxidizing the phosphite linkage of step (a) toform a phosphate linkage; and (d) removing the protecting group(“deprotection”) from the now substrate bound nucleoside coupled in step(a), to generate a reactive site for the next cycle of these steps. Thefunctionalized support (in the first cycle) or deprotected couplednucleoside (in subsequent cycles) provides a substrate bound moiety witha linking group for forming the phosphite linkage with a next nucleosideto be coupled in step (a). Final deprotection of nucleoside bases can beaccomplished using alkaline conditions such as ammonium hydroxide, in aknown manner.

[0004] The foregoing chemistry of the synthesis of polynucleotides isdescribed in detail, for example, in Caruthers, Science 230: 281-285,1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar etal., Nature 310: 105-110, 1984; and in “Synthesis of OligonucleotideDerivatives in Design and Targeted Reaction of OligonucleotideDerivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat.No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S.Pat. No. 5,869,643, EP 0294196, and elsewhere The phosphoramidite andphosphite triester approaches are most broadly used, but otherapproaches include the phosphodiester approach, the phosphotriesterapproach and the H-phosphonate approach. The substrates are typicallyfunctionalized to bond to the first deposited monomer. Suitabletechniques for functionalizing substrates with such linking moieties aredescribed, for example, in Southern, E. M., Maskos, U. and Elder, J. K.,Genomics, 13, 1007-1017, 1992.

[0005] In the case of array fabrication, different monomers may bedeposited at different addresses on the substrate during any oneiteration so that the different features of the completed array willhave different desired biopolymer sequences. One or more intermediatefurther steps may be required in each iteration, such as theconventional oxidation and washing steps in the case of in situfabrication of polynucleotide arrays.

[0006] In array fabrication, the quantities of polynucleotide availableare usually very small and expensive. Additionally, sample quantitiesavailable for testing are usually also very small and it is thereforedesirable to simultaneously test the same sample against a large numberof different probes on an array. These conditions require use of arrayswith large numbers of very small, closely spaced features. It isimportant in such arrays that features actually be present, that theyare put down accurately in the desired target pattern, are of thecorrect size, and that the DNA is uniformly coated within the feature.If any of these conditions are not met within a reasonable tolerance,and the array user is not aware of deviations outside such tolerance,the results obtained from a given array may be unreliable andmisleading. This of course can have serious consequences to diagnostic,screening, gene expression analysis or other purposes for which thearray is being used. However, in any system used to fabricate arrayswith the required small features, there is inevitably some degree oferror, either fixed (and hence repeated) and/or random. The presentinvention realizes that in the special case of in situ fabricationmethod or any other method requiring deposition of multiple droplets ata target feature location, drop deposition errors from cycle to cyclemay be different and are cumulative in determining errors in the finallyformed features.

[0007] It would be desirable then to provide a means by which errors infeatures resulting during an in situ or any array fabrication methodrequiring multiple droplet deposition for a target feature location, canbe readily determined.

SUMMARY OF THE INVENTION

[0008] The present invention further realizes that in array fabricationusing the in situ method or any method using multiple drop depositionfor a target feature location, the final error in a feature is a sum oferrors during respective droplet depositions, and that such individualdroplet errors may vary during each cycle at a given feature. Sucherrors may either be fixed or random. For example, in the fabrication ofa polynucleotide array using phosphoramidite chemistry, different dropdispensers (such as pulse jets which eject drops drops toward a surface)may be provided to deposit different phosphoramidite droplets. Eachnozzle may have its own inherent fixed drop dispensing error (such aserror in drop dispenser position within a head, droplet size, ordirection of drop). Also, the positioning system may have fixed inherenterrors. Further, random errors can occur which are different during thedispensing of any one droplet. For example, air currents may vary duringdifferent droplet dispensing steps, or ambient temperature variationsmay cause expansion/contraction in dispensing apparatus components whichaffects absolute and/or relative positions of dispensed droplets. Thus,an error in the final feature may be a sum of errors from individualdrop deposition at each feature. The present invention provides a meansof tracking such individual droplet deposition error for a feature andusing such multiple drop deposition error for that feature to determinean overall feature error.

[0009] In one aspect, the present invention provides a method whichincludes obtaining a set of multiple images of a target feature locationon an array of multiple features. Each image of the set represents thetarget feature location following deposition of a corresponding sub-setof multiple droplets for that feature. An overlay composite is generatedfrom the image set. Note that the processing for this can be done aseach image of a set is obtained or after all images of a set areobtained. By an “overlay composite” in this context is referenced thatinformation from at least one same location on the set of images iscombined or compared using some function (for example, logical AND orsome other operation). The same location may be, for example, all of thetarget feature location plus some predetermined area around thatlocation, or just some point or portion of the foregoing. A particularoverlay composite comprises a region of overlap of the multipledroplets. One or more feature characteristics (for example, the extent,size, shape or location of overlap of deposited droplets) may beprovided by the overlay composite. Typically, since an array willusually contain many features and it will be desirable to determinefeature characteristics of multiple such features (and perhaps all ofthem), the method may be used by obtaining multiple image sets ofrespective multiple target feature locations. In this case each imageset represents a corresponding target feature location with each imageof the set representing the location following deposition of acorresponding sub-set of multiple droplets for that feature. An overlaycomposite may then be generated from each of the image sets.

[0010] The above aspect may be used in the fabrication of arrays, or byan end user of the array or elsewhere. When the method is used infabrication, it will also typically include depositing multiple reagentdroplets for each of multiple target feature locations so as to formarray of features, and obtaining the image sets during such fabrication.In the fabrication, the image set is typically obtained by capturing animage of each target feature location following deposition of acorresponding drop sub-set (for example, from multiple captured imageseach of which simultaneously includes multiple target feature locationsfollowing a cycle of droplet depositions at those locations). When theabove aspect is used by an end user or elsewhere, the image sets may beobtained by other means (such as from a portable data storage mediumassociated with the array and carrying the image sets as digital data,or from communication with a remote array fabricator).

[0011] The overlay composite can be used for any of a number ofpurposes. For example, the overlay composite may also be used by thefabricator as a quality control tool such as by rejecting arrays withone or more feature characteristics which do not meet predeterminedrequirements, and/or by altering the deposition of additional dropletsfor the feature, or of droplets for other features on the same oranother array. Such rejecting or altering may be based at least in parton the overlay composite. Additionally, the overlay composite may beforwarded to a remote user of the array for use by them in interrogatingthe array or processing data resulting from the interrogation.Typically, the remote user will expose the array to a sample,interrogate the array following the exposure and optionally process theresults of the evaluation (such as for evaluating the samplecomposition, for example, for the presence of a component). Theinterrogation or processing may be based at least in part on the overlaycomposite. However, in an alternative aspect of the present invention,rather than (or in addition to) forwarding the composite overlay to theremote user, the image sets themselves may be stored on a storage mediumand forwarded to the remote user (such as by hard copy or on a portabledigital data storage medium, or by communication). Any forwarding to theremote user may be, for example, be by hard copy or portable digitaldata storage medium carrying the required information, or bycommunication over a suitable communication channel.

[0012] The present invention also provides an apparatus for fabricatingan array in accordance with any of the methods of the invention. Theapparatus may include a drop deposition system to deposit multiplereagent droplets for each of multiple target feature locations so as toform the array of features. An image capture system provides the one ormore sets of multiple images previously referenced. The apparatus mayfurther include either a processor which generates an overlay compositefrom the image set and optionally performs any the processing previouslydescribed, or a storage medium onto which the image set or an overlaycomposite of them are stored, or both. Optionally, a communicationmodule is present to communicate an image set or overlay composite to aremote location.

[0013] The present invention further provides a computer readablestorage medium carrying program code which can be used with an apparatusof the present invention, to cause it to execute the steps of a methodof the present invention.

[0014] The various aspects of the present invention can provide any oneor more of the following and/or other useful benefits. For example,individual droplet deposition errors during an in situ or any arrayfabrication method requiring multiple droplet deposition for a targetfeature location, can be readily determined and tracked, and resultantfeature errors determined from those individual droplet errors. Suchresults can be used as quality control tools during array fabricationand/or forwarded to a remote location for use in interrogating orprocessing interrogation results from the corresponding array.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 illustrates a substrate carrying multiple arrays, such asmay be fabricated by methods of the present invention;

[0016]FIG. 2 is an enlarged view of a portion of FIG. 1 showing multipleideal spots or features;

[0017]FIG. 3 is an enlarged illustration of a portion of the substratein FIG. 2;

[0018]FIGS. 4 and 5 illustrate examples of formation of overlaycomposites for less than ideal features;

[0019]FIG. 6 is a schematic diagram of a fabrication apparatus andmethod of the present invention; and

[0020]FIG. 7 is a schematic diagram of an apparatus at a user site whichcan execute a method of the present invention.

[0021] To facilitate understanding, identical reference numerals havebeen used, where practical, to designate identical elements that arecommon to the figures.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0022] In the present application, unless a contrary intention appears,the following terms refer to the indicated characteristics. A“biopolymer” is a polymer of one or more types of repeating units.Biopolymers are typically found in biological systems and particularlyinclude peptides or polynucleotides, as well as such compounds composedof or containing amino acid analogs or non-amino acid groups, ornucleotide analogs or non-nucleotide groups. This includespolynucleotides in which the conventional backbone has been replacedwith a non-naturally occurring or synthetic backbone, and nucleic acids(or synthetic or naturally occurring analogs) in which one or more ofthe conventional bases has been replaced with a group (natural orsynthetic) capable of participating in Watson-Crick type hydrogenbonding interactions. Polynucleotides include single or multiplestranded configurations, where one or more of the strands may or may notbe completely aligned with another. A “nucleotide” refers to a sub-unitof a nucleic acid and has a phosphate group, a 5 carbon sugar and anitrogen containing base, as well as functional analogs (whethersynthetic or naturally occurring) of such sub-units which in the polymerform (as a polynucleotide) can hybridize with naturally occurringpolynucleotides in a sequence specific manner analogous to that of twonaturally occurring polynucleotides. For example, a “biopolymer”includes DNA (including cDNA), RNA, oligonucleotides, and PNA and otherpolynucleotides as described in U.S. Pat. No. 5,948,902 and referencescited therein (all of which are incorporated herein by reference),regardless of the source. An “oligonucleotide” generally refers to anucleotide multimer of about 10 to 100 nucleotides in length, while a“polynucleotide” includes a nucleotide multimer having any number ofnucleotides. A “biomonomer” references a single unit, which can belinked with the same or other biomonomers to form a biopolymer (forexample, a single amino acid or nucleotide with two linking groups oneor both of which may have removable protecting groups). A biomonomerfluid or biopolymer fluid reference a liquid containing either abiomonomer or biopolymer, respectively (typically in solution).

[0023] An “array”, unless a contrary intention appears, includes any oneor two dimensional arrangement of addressable regions bearing aparticular chemical moiety to moieties (for example, biopolymers such aspolynucleotide sequences) associated with that region. An array is“addressable” in that it has multiple regions of different moieties (forexample, different polynucleotide sequences) such that a region (a“feature” or “spot” of the array) at a particular predetermined location(an “address”) on the array will detect a particular target or class oftargets (although a feature may incidentally detect non-targets of thatfeature). Array features are typically, but need not be, separated byintervening spaces. In the case of an array, the “target” will bereferenced as a moiety in a mobile phase (typically fluid), to bedetected by probes (“target probes”) which are bound to the substrate atthe various regions. However, either of the “target” or “target probes”may be the one which is to be evaluated by the other (thus, either onecould be an unknown mixture of polynucleotides to be evaluated bybinding with the other). An “array layout” refers collectively to one ormore characteristics of the features, such as feature positioning, oneor more feature dimensions, and some indication of a moiety at a givenlocation. “Hybridizing” and “binding”, with respect to polynucleotides,are used interchangeably.

[0024] When one item is indicated as being “remote” from another, thisis referenced that the two items are at least in different buildings,and may be at least one mile, ten miles, or at least one hundred milesapart. “Communicating” information references transmitting the datarepresenting that information over a suitable communication channel (forexample, a private or public network). “Forwarding” an item refers toany means of getting that item from one location to the next, whether byphysically transporting that item and includes, at least in the case ofdata, physically transporting a medium carrying the data orcommunicating the data. An array “package” may be the array plus only asubstrate on which the array is deposited, although the package mayinclude other features (such as a housing with a chamber). A “chamber”references an enclosed volume (although a chamber may be accessiblethrough one or more ports). It will also be appreciated that throughoutthe present application, that words such as “top”, “upper”, and “lower”are used in a relative sense only. “Fluid” is used herein to reference aliquid. An “image” in relation to a target feature location followingdroplet deposition, refers to data on shape, size or position of thewhole or part of the droplet (in liquid form or after drying),regardless of how such data was obtained (for example by capturing witha camera, or by scanning with a laser beam, or by some other means). A“set” or a “sub-set” may have one or more members (for example, one ormore droplets). Reference to a singular item, includes the possibilitythat there are plural of the same items present. All patents and othercited references are incorporated into this application by reference.

[0025] Referring first to FIGS. 1-3, typically methods and apparatus ofthe present invention generate or use a contiguous planar substrate 10carrying one or more arrays 12 disposed across a front surface 11 a ofsubstrate 10 and separated by inter-array areas 13. A back side 11 b ofsubstrate 10 does not carry any arrays 12. The arrays on substrate 10can be designed for testing against any type of sample, whether a trialsample, reference sample, a combination of them, or a known mixture ofpolynucleotides (in which latter case the arrays may be composed offeatures carrying unknown sequences to be evaluated). Each array 12 hasassociated with it a unique identification in the form of a bar code356. While ten arrays 12 are shown in FIG. 1 and the differentembodiments described below may use substrates with particular numbersof arrays, it will be understood that substrate 10 and the embodimentsto be used with it, may use any number of desired arrays 12. Similarly,substrate 10 may be of any shape, and any apparatus used with it adaptedaccordingly. Depending upon intended use, any or all of arrays 12 may bethe same or different from one another and each will contain multiplespots or features 16 of biopolymers in the form of polynucleotides. Atypical array may contain from more than ten, more than one hundred,more than one thousand or ten thousand features, or even more than fromone hundred thousand features. All of the features 16 may be different,or some or all could be the same. In the embodiment illustrated, thereare interfeature areas 17 between features, which do not carry anypolynucleotide. It will be appreciated though, that the interfeatureareas 17 could be of various sizes and configurations. It will beappreciated that there need not be any space separating arrays 12 fromone another, nor features 16 within an array from one another. However,in the case where arrays 12 are formed by the conventional in situmethod as described above, by depositing for each feature a droplet ofreagent in each step such as by using a pulse jet such as an inkjet typehead, such interfeature areas 17 will typically be present. Each featurecarries a predetermined polynucleotide (which includes the possibilityof mixtures of polynucleotides). As per usual, A, C, G, T represent theusual nucleotides. It will be understood that there is usually a linkermolecule (not shown) of any known types between the front surface 11 aand the first nucleotide.

[0026]FIGS. 2 and 3 illustrate ideal features where the actual featuresformed are the same as the target (or “aim”) features, with each feature16 being uniform in shape, size and composition, and the features beingregularly spaced. Such an array when fabricated by the in situ method,would require all reagent droplets for each feature to be uniform inshape and accurately deposited at the target feature location. Inpractice, such an ideal result is difficult to obtain due to both thefixed and random errors such as those discussed above. FIG. 4 showsimages of two phosphoramidite droplets deposited in different cycles,for a particular feature (that is FIG. 4 shows an image set for thatfeature), and illustrates a less than ideal result. FIG. 5 similarlyillustrates an image set for another feature of five phosphoramiditedroplets with a less than ideal actual feature being formed (referenceto the shape of the deposited droplets will, in the followingdiscussion, refer to dried droplet shape). The droplet errorsillustrated in FIGS. 4 and 5 have been somewhat exaggerated for thepurpose of clarity. For the discussion which follows, it will be assumedthat each “sub-set” of droplets after which an image is captured,consists of only one set. However, in practice each such sub-set willtypically consist of multiple droplets of the same composition. In FIG.4, a nucleoside phosphoramidite reagent droplet 404 (such droplets beingoften referenced simply as “phosphoramidite” droplets) of circular shapehas been deposited in one cycle with its center exactly at the center402 of the target feature location. However, another phosphoramiditedroplet 400 deposited in a subsequent or earlier cycle has an ellipticalshape due to fixed or random errors, and has its center mis-aligned withtarget feature center 402. If the target feature was only to be formedfrom the foregoing two phosphoramidites, the actual feature having thedesired nucleotide sequence would only consist of overlap region 406(which is the intersection of the droplets, or logical AND of them),which is neither of the target circular shape nor on the target featurecenter. Similarly, in FIG. 5 where five phosphoramidites droplets 410,416, 418, 422 are deposited in different cycles for a target feature,one droplet 410 is centered on target feature center 412 and iscorrectly shaped (circular) but is too large, while droplets 416, 418have the target shape and size but are off the center 412, and droplet422 is both of the wrong shape, size and position. In this case, theresulting actual feature 430 of five nucleotides in length, would againbe the overlap (or logical AND) of the droplets. Note that actualfeature 430 is of incorrect shape and size, and does not even overlapthe target center 412.

[0027] Thus, as illustrated in FIGS. 4 and 5, when an image of each ofthe deposited reagent droplets in the multi-cycle in situ synthesis areobtained (such as by obtaining an image of each entire droplet),formation of an overlay composite can provide information on one or morecharacteristics of an actual feature. In the particular cases describedin connection with FIGS. 4 and 5, a logical AND operation is applied tothe set of such images for a given feature to provide the overlap regioncharacteristics (such as size, shape and location) and hence thecharacteristics of the actual feature. As is apparent from FIGS. 4 and5, overlay composites formed from a logical AND operation, automaticallyprovides an evaluation of feature characteristics.

[0028] Referring now to FIG. 6, an apparatus of the present inventionwhich can execute a method of the present invention, will now bedescribed.

[0029] Referring now to FIG. 6 the apparatus shown includes a substratestation 20 on which can be mounted a substrate 10. Pins or similar means(not shown) can be provided on substrate station 20 by which toapproximately align substrate 10 to a nominal position thereon.Substrate station 20 can include a vacuum chuck connected to a suitablevacuum source (not shown) to retain a substrate 14 without exerting toomuch pressure thereon, since substrate 14 is often made of glass. Aflood station 68 is provided which can expose the entire surface ofsubstrate 10, when positioned beneath station 68 as illustrated inbroken lines in FIG. 4, to a fluid typically used in the in situprocess, and to which all features must be exposed during each cycle(for example, oxidizer, deprotection agent, and wash buffer).

[0030] A dispensing head 210 is retained by a head retainer 208. Thepositioning system includes a carriage 62 connected to a firsttransporter 60 controlled by processor 140 through line 66, and a secondtransporter 100 controlled by processor 140 through line 106.Transporter 60 and carriage 62 are used execute one axis positioning ofstation 20 (and hence mounted substrate 10) facing the dispensing head210, by moving it in the direction of arrow 63, while transporter 100 isused to provide adjustment of the position of head retainer 208 (andhence head 210) in a direction of axis 204. In this manner, head 210 canbe scanned line by line, by scanning along a line over substrate 10 inthe direction of axis 204 using transporter 100, while line by linemovement of substrate 10 in a direction of axis 63 is provided bytransporter 60. Transporter 60 can also move substrate holder 20 toposition substrate 10 beneath flood station 68 (as illustrated by thesubstrate 10 shown in broken lines in FIG. 4). Head 210 may alsooptionally be moved in a vertical direction 202, by another suitabletransporter (not shown). It will be appreciated that other scanningconfigurations could be used. It will also be appreciated that bothtransporters 60 and 100, or either one of them, with suitableconstruction, could be used to perform the foregoing scanning of head210 with respect to substrate 10. Thus, when the present applicationrecites “positioning” one element (such as head 210) in relation toanother element (such as one of the stations 20 or substrate 10) it willbe understood that any required moving can be accomplished by movingeither element or a combination of both of them. The head 210, thepositioning system, and processor 140 together act as the depositionsystem of the apparatus. An encoder 30 communicates with processor 140to provide data on the exact location of substrate station 20 (and hencesubstrate 10 if positioned correctly on substrate station 20), whileencoder 34 provides data on the exact location of holder 208 (and hencehead 210 if positioned correctly on holder 208). Any suitable encoder,such as an optical encoder, may be used which provides data on linearposition.

[0031] Processor 140 also has access through a communication module 144to a communication channel 180 to communicate with a remote station.Communication channel 180 may, for example, be a Wide Area Network(“WAN”), telephone network, satellite network, or any other suitablecommunication channel.

[0032] Head 210 may be of a type commonly used in an ink jet type ofprinter and may, for example, include five or more chambers (at leastone for each of four nucleoside phosphoramidite monomers plus at leastone for an activator solution) each communicating with a correspondingset of multiple drop dispensing orifices and multiple ejectors which arepositioned in the chambers opposite respective orifices. Each ejector isin the form of an electrical resistor operating as a heating elementunder control of processor 140 (although piezoelectric elements could beused instead). Each orifice with its associated ejector and portion ofthe chamber, defines a corresponding pulse jet. It will be appreciatedthat head 210 could, for example, have more or less pulse jets asdesired (for example, at least ten or at least one hundred pulse jets).Application of a single electric pulse to an ejector will cause adroplet to be dispensed from a corresponding orifice. Certain elementsof the head 210 can be adapted from parts of a commercially availablethermal inkjet print head device available from Hewlett-Packard Co. aspart no. HP51645A. Alternatively, multiple heads could be used insteadof a single head 210, each being similar in construction to head 210 andbeing provided with respective transporters under control of processor140 for independent movement. In this alternate configuration, each headmay dispense a corresponding biomonomer (for example, one of fournucleoside phosphoramidites) or an activator solution.

[0033] As is well known in the ink jet print art, the amount of fluidthat is expelled in a single activation event of a pulse jet, can becontrolled by changing one or more of a number of parameters, includingthe orifice diameter, the orifice length (thickness of the orificemember at the orifice), the size of the deposition chamber, and the sizeof the heating element, among others. The amount of fluid that isexpelled during a single activation event is generally in the rangeabout 0.1 to 1000 pL, usually about 0.5 to 500 pL and more usually about1.0 to 250 pL. A typical velocity at which the fluid is expelled fromthe chamber is more than about 1 m/s, usually more than about 10 m/s,and may be as great as about 20 m/s or greater. As will be appreciated,if the orifice is in motion with respect to the receiving surface at thetime an ejector is activated, the actual site of deposition of thematerial will not be the location that is at the moment of activation ina line-of-sight relation to the orifice, but will be a location that ispredictable for the given distances and velocities.

[0034] The apparatus can deposit droplets to provide features which mayhave widths (that is, diameter, for a round spot) in the range from aminimum of about 10 μm to a maximum of about 1.0 cm. In embodimentswhere very small spot sizes or feature sizes are desired, material canbe deposited according to the invention in small spots whose width is inthe range about 1.0 μm to 1.0 mm, usually about 5.0 μm to 500 μm, andmore usually about 10 μm to 200 μm.

[0035] The apparatus further includes a display 310, speaker 314, andoperator input device 312. Operator input device 312 may, for example,be a keyboard, mouse, or the like. Processor 140 has access to a memory141, and controls print head 210 (specifically, the activation of theejectors therein), operation of the positioning system, operation ofeach jet in print head 210, and operation of display 310 and speaker314. Memory 141 may be any suitable device in which processor 140 canstore and retrieve data, such as magnetic, optical, or solid statestorage devices (including magnetic or optical disks or tape or RAM, orany other suitable device, either fixed or portable). Processor 140 mayinclude a general purpose digital microprocessor suitably programmedfrom a computer readable medium carrying necessary program code, toexecute all of the steps required by the present invention, or anyhardware or software combination which will perform those or equivalentsteps. The programming can be provided remotely to processor 141, orpreviously saved in a computer program product such as memory 141 orsome other portable or fixed computer readable storage medium using anyof those devices mentioned below in connection with memory 141. Forexample, a magnetic or optical disk 324 a may carry the programming, andcan be read by disk writer/reader 326.

[0036] A writing system which is under the control of processor 140,includes a writer in the form of a printer 150 which can writeidentifications onto substrate 10 by printing them in the form of thebar codes 356 (or alternatively on a housing carrying the substrate)each in association with a corresponding array 12 as shown in FIG. 1.Printer 150 may accomplish this task before or after formation of thearray by the drop deposition system. The writing system further includesa data writer/reader 326 (such as an optical or magnetic disk drive)which can write data to a portable computer readable storage medium(such as an optical or magnetic disk). A cutter 152 is provided to cutsubstrate 10 into individual array units 15 each carrying acorresponding array 12 and bar code 356. An illumination source 170 candirect light through a beam splitter in the form of dichroic mirror 172to illuminate substrate 10 on station 20 from below (the light shiningthrough the back side 11 b of substrate 10). An image capture system inthe form of linescan camera 174 and processor 140, captures imagesthrough mirror 172 each of which simultaneously includes multiple targetfeature locations on substrate 10 following a cycle of dropletdepositions at those locations. Processor 140 can extract from suchcaptured images, the image set for each target feature location plussome predetermined region around that location (for example, a squaresurrounding that location) images each of which simultaneously includesmultiple target feature locations following a cycle of dropletdepositions at those locations. Camera 174 may have a pixel resolutionof 30 μm or less, or 20 μm or less, and more preferably 10 μm or less.Any suitable camera may be used such as a CCD camera capturing arectangular image, or a linescan camera. Processor 140 coordinatescapture of the lines with the movement of substrate 10 during depositionof the reagent droplets.

[0037] The above described components in FIG. 6 represent an apparatusfor producing an addressable array, which is sometimes referenced hereinas a “fabrication station”. FIG. 7 illustrates an apparatus forreceiving an addressable array, in particular a single “user station”,which is remote from the fabrication station. The user station includesa processor 162, a memory 184, a scanner 160 which can interrogate anarray, data writer/reader 186 (which may be capable of writing/readingto the same type of media as writer/reader 320), and a communicationmodule 164 which also has access to communication channel 180. Memory184 can be any type of memory such as those used for memory 141. Scanner160 can be any suitable apparatus for interrogating an array, such asone which can read the location and intensity of fluorescence at eachfeature of an array following exposure to a fluorescently labeledsample. For example, such a scanner may be similar to the GENEARRAYscanner available from Hewlett-Packard, Palo Alto, Calif. Scanner 160also includes though, a first bar code reader to read a each bar code356 appearing on segment 15.

[0038] It will be understood that there may be multiple such userstations, each remote from the fabrication station and each other, inwhich case the fabrication station acts as a central fabrication station(that is, a fabrication station which services more than one remote userstation at the same or different times). One or more such user stationsmay be in communication with the fabrication station at any given time.It will also be appreciated that processors 140 and 162 can beprogrammed from any computer readable medium carrying a suitablecomputer program. For example, such a medium can be any memory devicesuch as those described in connection with memory 141, and may be readlocally (such as by reader/writer 320 in the case of processor 140 orwriter/reader 186 in the case of processor 162) or from a remotelocation through communication channel 180.

[0039] The operation of the fabrication station will now be described.It will be assumed that a substrate 10 on which arrays 12 are to befabricated, is in position on station 20 and that processor 140 isprogrammed with the necessary layout information to fabricate targetarrays 12. Processor 140 will generate a unique identification for eacharray 12 in the form of a corresponding bar code 356. Each identifiermay be stored in memory 141 in association with the corresponding imagesets for each feature of an array 12, or with composite overlays derivedfrom those sets by processor 140 (as mentioned below).

[0040] Processor 140 controls fabrication, as described above, togenerate the one or more arrays on substrate 10 by depositing for eachtarget feature, multiple droplets of phosphoramidite reagents, and sendssubstrate 10 to flood station 68 for intervening or final steps asrequired, all in accordance with the conventional in situ polynucleotidearray fabrication process described above. Preferably at each feature,during any one cycle multiple droplets of both the same phorsphoramiditesolution and a tetrazole activator are deposited. For example, aboutfive droplets of each may be deposited (making for a total of tendroplets in each cycle). Camera 174 captures images through mirror 172each of which simultaneously includes multiple target feature locationson substrate 10 following a cycle of droplet depositions at thoselocations. The sub-set of droplets after which each image can becaptured can equal this total number. However, it will be appreciatedthat this need not be the case (for example, ten total droplets may bedeposited during a cycle, with an image being captured after eachsub-set of five droplets, which two images are processed to determinetotal deposition area with a logical OR). Processor 140 extracts fromsuch captured images, the image set for each target feature locationplus some predetermined region around that location. At this point, ifit is desired to provide feature characteristic information to an arrayuser, processor 140 may store such image sets for all features of anarray 12 into memory 141, or more preferably generates an overlaycomposite from all the sets (preferably by a logical AND operation asdescribed above) and stores the overlay composites for all the featuresof an array 12 into memory 141. In either event, the image sets oroverlay composites for an array 12 are stored in association with thecorresponding unique identifier for that array. Note that the generationof an overlay composite can be performed after all images of a set arecollected, or data processing can be performed as the images areobtained (that is, each image is processed as required immediately afterit is obtained). Optionally, the overlay composites for an array or theimage sets themselves, can later be stored onto a portable storagemedium 324 b in association with the corresponding array uniqueidentification, by writer/reader 326 for provision to a remote user.Note that the overlay composites effectively represent a map of actualfeatures on the array. It will be understood that the data form of theoverlay composites can be varied though, such as being absolute data orrelative data (such as relating the actual feature map to acorresponding target feature map forwarded from the fabricationstation).

[0041] If it is not desired to provide overlay composites to an arrayuser, but only to use the overlay composites as a quality controlmeasure, then they can be used to reject an array 12 and direct ittoward a garbage bin (not shown) if one or more predetermined number offeatures have one or more characteristics outside a predeterminedtolerance. Such characteristics may include a minimum feature size (notethat a feature size would be considered zero where the images indicate acomplete failure to deposit a required phosphoramidite reagent duringany one cycle). In an alternative, the deposition of additional dropletsfor a selected feature or droplets deposited at other features on thesame or another array, can be based at least in part on an evaluatedcharacteristic from an overlay composite for other droplets for thatselected or other features. For example, if processor 140 generateslogical AND for image sets of all features in an array and determinesthe resulting features have a characteristic outside a predeterminedlimit, such as a common mispositioning error, processor 140 may be ableto compensate for this by repositioning head 210 during fabrication ofother arrays. Other functions can be used, such as a logical “OR” whereall of the area which has been covered by the various deposition cyclescan be determined. The result of a logical “OR” shows the region whereboth full-length and non full-length polynucleotides are obtained (whichcan be helpful with, for example, HPLC analysis of DNA fragments)

[0042] It will be appreciated also, and any of the foregoing uses of theoverlay composites can be combined. For example overlay composites canbe both used to reject an array as described above, and the overlaycomposites or image sets still stored and forwarded to an end user forarrays that were not rejected. Furthemore, information from the imagesets can be used for other purposes, such as monitoring variation in thesize of a deposited droplet sub-set (which may indicate a problem).

[0043] Either before array fabrication on substrate 10 has beencommenced, or after it has been completed, substrate 10 may be sent towriter 150 which, under control of processor 140, writes the unique barcodes 356 on substrate 10 each in association with its correspondingarray (by being physically close to it in the manner shown in FIG. 1).The substrate 10 is then sent to a cutter 152 wherein portions ofsubstrate 10 carrying an individual array 12 and its associated localidentifier 356 are separated from the remainder of substrate 10, toprovide multiple array units 15. The array unit 15 is placed in package340 with storage medium 324 b (if used) carrying overlay composites orimage sets for the corresponding array 12, and the package then shippedto a remote user station.

[0044] The above sequence can be repeated at the fabrication station asdesired for multiple substrates 10 in turn. As mentioned above, thefabrication station may act as a central fabrication station for each ofmultiple remote user stations, in the same manner as described above.Whether or not the fabrication station acts as a central fabricationstation, it can optionally maintain a database of unique arrayidentifiers in memory 141, each in association with the correspondingimage sets or overlay composites.

[0045] At the user station of FIG. 7, the resulting package 340 is thenreceived from the remote fabrication station. A sample, for example atest sample, is exposed to the array 12 on the array unit 15 received inpackage 340. Following hybridization and washing in a known manner, thearray is then inserted into scanner 160 and interrogated by it to obtaininterrogation results (such as information representing the fluorescencepattern on the array 12). The first reader in scanner 160 also reads theidentifier 356 present on the array substrate 10 in association with thecorresponding array 12. Using identifier 356, processor 162 may thenobtain the corresponding overlay composites or image sets for array 12from portable storage medium 324 b or from the database of suchinformation in memory 141 by communicating with that database throughcommunication module 164 and communication channel 180. Alternatively,in the case where processor 162 has only obtained image sets, it cangenerate an overlay composite itself as needed.

[0046] Once processor 162 has obtained the overlay composites or imagesets for an array 12, it can then control interrogation of thecorresponding array by scanner 160 and/or the processing of the resultsof the interrogation, using such information. For example, an overlaycomposite may indicate a specific feature of such low quality (that is,having a characteristic outside a predetermined range) which isredundant with another feature, such that the scanner need notinterrogate that specific feature for a given test, or alternativelythat information read from that address can be ignored or deleted asuseless. Following array interrogation, further processing of theinterrogation results can be performed to evaluate the samplecomposition (such as for a specific target) based on the results of theinterrogation. This further processing can be done by processor 162 orby a user examining the interrogation results. The interrogation results(raw data) or processed results from the interrogation results, could beforwarded (such as by communication) to a remote location for furtherevaluation and/or processing, or use, using communication channel 180 orreader/writer 186 and medium 190. This data may be transmitted by othersas required to reach the remote location, or re-transmitted to elsewhereas desired.

[0047] In a variation of the above, it is possible that each array 12and its substrate 10 may be contained with a suitable housing. Such ahousing may include a closed chamber accessible through one or moreports normally closed by septa, which carries the substrate 10. It willalso be appreciated that an image of a target feature location mayinclude just a portion of that location, and that an image setpreferably, but not necessarily, includes all of the droplets for thatfeature.

[0048] Modifications in the particular embodiments described above are,of course, possible. For example, where a pattern of arrays is desired,any of a variety of geometries may be constructed other than theorganized rows and columns of arrays 12 of FIG. 1. For example, arrays12 can be arranged in a series of curvilinear rows across the substratesurface (for example, a series of concentric circles or semi-circles ofspots), and the like. Similarly, the pattern of regions 16 may be variedfrom the organized rows and columns of spots in FIG. 2 to include, forexample, a series of curvilinear rows across the substrate surface(forexample, a series of concentric circles or semi-circles of spots), andthe like. Even irregular arrangements of the arrays or the regionswithin them can be used.

[0049] The present methods and apparatus may be used to depositbiopolymers or other moieties on surfaces of any of a variety ofdifferent substrates, including both flexible and rigid substrates.Preferred materials provide physical support for the deposited materialand endure the conditions of the deposition process and of anysubsequent treatment or handling or processing that may be encounteredin the use of the particular array. The array substrate may take any ofa variety of configurations ranging from simple to complex. Thus, thesubstrate could have generally planar form, as for example a slide orplate configuration, such as a rectangular or square or disc. In manyembodiments, the substrate will be shaped generally as a rectangularsolid, having a length in the range about 4 mm to 200 mm, usually about4 mm to 150 mm, more usually about 4 mm to 125 mm; a width in the rangeabout 4 mm to 200 mm, usually about 4 mm to 120 mm and more usuallyabout 4 mm to 80 mm; and a thickness in the range about 0.01 mm to 5.0mm, usually from about 0.1 mm to 2 mm and more usually from about 0.2 to1 mm. However, larger substrates can be used, particularly when such arecut after fabrication into smaller size substrates carrying a smallertotal number of arrays 12. Substrates of other configurations andequivalent areas can be chosen. The configuration of the array may beselected according to manufacturing, handling, and use considerations.

[0050] The substrates may be fabricated from any of a variety ofmaterials. In certain embodiments, such as for example where productionof binding pair arrays for use in research and related applications isdesired, the materials from which the substrate may be fabricated shouldideally exhibit a low level of non-specific binding during hybridizationevents. In many situations, it will also be preferable to employ amaterial that is transparent to visible and/or UV light. For flexiblesubstrates, materials of interest include: nylon, both modified andunmodified, nitrocellulose, polypropylene, and the like, where a nylonmembrane, as well as derivatives thereof, may be particularly useful inthis embodiment. For rigid substrates, specific materials of interestinclude: glass; fused silica, silicon, plastics (for example,polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof, and the like); metals (for example, gold, platinum, andthe like).

[0051] The substrate surface onto which the polynucleotide compositionsor other moieties is deposited may be porous or non-porous, smooth orsubstantially planar, or have irregularities, such as depressions orelevations. The surface may be modified with one or more differentlayers of compounds that serve to modify the properties of the surfacein a desirable manner. Such modification layers, when present, willgenerally range in thickness from a monomolecular thickness to about 1mm, usually from a monomolecular thickness to about 0.1 mm and moreusually from a monomolecular thickness to about 0.001 mm. Modificationlayers of interest include: inorganic and organic layers such as metals,metal oxides, polymers, small organic molecules and the like. Polymericlayers of interest include layers of: peptides, proteins, polynucleicacids or mimetics thereof (for example, peptide nucleic acids and thelike); polysaccharides, phospholipids, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, and the like, wherethe polymers may be hetero- or homopolymeric, and may or may not haveseparate functional moieties attached thereto (for example, conjugated),

[0052] Various further modifications to the particular embodimentsdescribed above are, of course, possible. Accordingly, the presentinvention is not limited to the particular embodiments described indetail above.

What is claimed is:
 1. A method comprising: obtaining a set of multipleimages of a target feature location on an array of multiple features,each image of the set representing the target feature location followingdeposition of a corresponding sub-set of multiple droplets for thatfeature; and generating an overlay composite from the image set.
 2. Amethod according to claim 1 wherein the overlay composite comprises aregion of overlap of the droplet sub-sets.
 3. A method according toclaim 1 wherein: multiple image sets are obtained of respective multipletarget feature locations, each image set representing a correspondingtarget feature location and in which each image represents the locationfollowing deposition of a corresponding droplet sub-set for thatfeature; and an overlay composite is generated from each of the imagesets.
 4. A method of fabricating an array of features, comprising:depositing multiple reagent droplets for each of multiple target featurelocations so as to form the array of features; obtaining a set ofmultiple images of a target feature location, each image of the setrepresenting the target feature location following deposition of acorresponding sub-set of multiple droplets for that feature; andgenerating an overlay composite from the image set.
 5. A methodaccording to claim 4 wherein the multiple reagent droplets are ejectedfrom a pulse-jet.
 6. A method according to claim 4 wherein the overlaycomposite comprises a region of overlap of the droplet sub-sets.
 7. Amethod according to claim 4 wherein multiple image sets are obtained ofrespective multiple target feature locations, each image setrepresenting a corresponding target feature location and in which eachimage represents the location following deposition of a correspondingsub-set of multiple droplets for that feature.
 8. A method according toclaim 7 wherein each set is obtained from multiple captured images eachof which simultaneously includes multiple target feature locationsfollowing a cycle of droplet depositions at those locations.
 9. A methodaccording to claim 4 wherein the array is a biopolymer array.
 10. Amethod according to claim 4 wherein the array is a polynucleotide arrayand the set of images includes, for all nucleotide droplets for thefeature, an image representing the target feature location followingdeposition of all droplets for a corresponding one of the nucleotidemonomers.
 11. A method according to claim 4 additionally comprisingeither altering the deposition of additional droplets for the feature,or of droplets for other features on the same or another array, based atleast in part on the overlay composite.
 12. A method according to claim4 wherein the overlay composite is stored on a storage medium andforwarded to a remote user of the array.
 13. A method of fabricating anarray of features, comprising: depositing multiple reagent droplets foreach of multiple target feature locations so as to form the array offeatures; obtaining a set of multiple images of a target featurelocation, each image of the set representing the target feature locationfollowing deposition of a corresponding sub-set of multiple droplets forthat feature; and storing the set on a storage medium.
 14. A methodaccording to claim 13 wherein the multiple droplets are deposited from apulse-jet.
 15. A method according to claim 13 wherein the set is storedon a storage medium and forwarded to a remote user of the array.
 16. Amethod according to claim 1 additionally comprising: exposing the arrayto a sample; interrogating the array following the exposure andoptionally processing results of the interrogation; wherein either theinterrogation or processing is based at least in part on the overlaycomposite.
 17. A method according to claim 15 wherein a result of theinterrogation or processing is forwarded to a remote location.
 18. Amethod comprising transmitting data representing a result of theinterrogation or processing from the method of claim
 17. 19. Anapparatus for fabricating an array of features, comprising: a dropdeposition system to deposit multiple reagent droplets for each ofmultiple target feature locations so as to form the array of features;an image capture system which provides a set of multiple images of atarget feature location, each image of the set representing the targetfeature location following deposition of a corresponding sub-set ofmultiple droplets for that feature; and a processor which generates anoverlay composite from the image set.
 20. An apparatus according toclaim 19 wherein the drop deposition system comprises a pulse-jet. 21.An apparatus according to claim 19 wherein the overlay compositegenerated by the processor comprises a region of overlap of the dropletsub-sets.
 22. An apparatus according to claim 19 wherein the imagecapture system provides multiple image sets of respective multipletarget feature locations, each image set representing a correspondingtarget feature location and in which each image represents the locationfollowing deposition of a corresponding sub-set of multiple droplets forthat feature.
 23. An apparatus according to claim 22 wherein the imagecapture system provides each set from multiple captured images each ofwhich simultaneously includes multiple target feature locationsfollowing a cycle of droplet depositions at those locations.
 24. Anapparatus according to claim 19 wherein the array is a polynucleotidearray and the set of images includes, for multiple nucleotide reagentdroplets for the feature, an image representing the target featurelocation following deposition of a corresponding one of the nucleotidemonomer droplets.
 25. An apparatus according to claim 19 wherein thedrop deposition system alters the deposition of additional droplets forthe feature, or of droplets for other features on the same or anotherarray, based at least in part on the overlay composite.
 26. An apparatusaccording to claim 19 additionally comprising a storage medium and acommunication module, wherein the processor stores the overlay compositeon the storage medium and causes the communication module to communicatethe stored overlay composite to a remote user of the array.
 27. Anapparatus for fabricating an array of features, comprising: a dropdeposition system to deposit multiple reagent droplets for each ofmultiple target feature locations so as to form the array of features;an image capture system which provides a set of multiple images of atarget feature location, each image of the set representing the targetfeature location following deposition of a corresponding sub-set ofmultiple droplets for that feature; and a storage medium onto which theimage set or an overlay composite of them are stored.
 28. An apparatusaccording to claim 27, additionally comprising a communication module tocommunicate the stored image set or an overlay composite to a remoteuser of the array.
 29. A computer program product comprising a computerreadable storage medium carrying computer readable program code, for usewith an apparatus for fabricating an array of features which apparatusincludes a drop deposition system and an image capture system under thecontrol of a computer, the program code when loaded into the computerperforming the steps of: depositing multiple reagent droplets from thedrop deposition system for each of multiple target feature locations, soas to form an array of features; obtaining from the image capturesystem, a set of multiple images of a target feature location on anarray of multiple features, each image of the set representing thetarget feature location following deposition of a corresponding sub-setof multiple droplets for that feature; and generating in the computer anoverlay composite from the image set.
 30. A computer program productcomprising a computer readable storage medium carrying computer readableprogram code, for use with an apparatus having a data retrieval unitunder the control of a computer, the program code when loaded into thecomputer performing the steps of: obtaining through the data retrievalunit a set of multiple images of a target feature location on an arrayof multiple features, each image of the set representing the targetfeature location following deposition of a corresponding sub-set ofmultiple droplets for that feature; and generating in the computer anoverlay composite from the image set.
 31. A computer program productaccording to claim 30 wherein the program code generates an overlaycomposite comprising a region of overlap of the multiple droplets.
 32. Acomputer program product according to claim 30 wherein: multiple imagesets are obtained of respective multiple target feature locations, eachimage set representing a corresponding target feature location and inwhich each image represents the location following deposition of acorresponding sub-set of multiple droplets for that feature; and anoverlay composite is generated in the computer from each of the imagesets.
 33. A computer program product according to claim 30 wherein theprogram code additionally either alters the deposition of additionaldroplets for the feature, or of droplets for other features on the sameor another array, based at least in part on the overlay composite.
 34. Acomputer program product according to claim 30 wherein the program codeadditionally performs the step of: controlling at least in part,interrogation of the array following exposure to a sample; andoptionally processing results of the interrogation; wherein either theinterrogation or processing is based at least in part on the overlaycomposite.